1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis.
2. Summary of the Related Art
Oligonucleotides have become indispensible tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g.,Methods in Molecular Biology, Vol 20: Protocols for oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6, 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72, 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34, 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22, 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28, 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23, 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager el al., Biochemistry 27, 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Antl. Acad. Sci. USA 85, 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
Solid phase synthesis of oligonucleotides by each of the foregoing methods involves the same generalized protocol. Briefly, this approach comprises anchoring the 3xe2x80x2-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Desired internucleoside linkages are formed between the 3xe2x80x2 functional group of the incoming nucleoside and the 5xe2x80x2 hydroxyl group of the 5xe2x80x2-most nucleoside of the nascent, support-bound oligonucleotide.
Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 umol to 1 mmol and higher). See Padmapriya et al., Antisense Res. Dev. 4, 185 (1994). Several modifications of the standard phosphoramidite methods have already been reported to facilitate the synthesis (Padmapriya et al., supra; Ravikumar et al., Tetrahedron 50, 9255 (1994); Theisen et al., Nucleosides and Nucleotides 12, 43 (1994); and Iyer et al., Nucleosides and Nucleotides 14, 1349 (1995)) and isolation (Kuijpers et al. Nucl. Acids Res. 18, 5197 (1990); and Reddy et al., Tetrahedron Lett. 35, 4311 (1994)) of oligonucleotides.
The routine synthesis of oligonucleotides is presently carried out using various N-acyl protecting groups for the nucleoside bases, such as isobutyryl (for guanine), and benzoyl for adenine and cytosine. After the synthesis of the oligonucleotides is carried out using either phosphoramidite chemistry or H-phosphonate chemistry, the protecting groups are removed by treatment with ammonia at 55-60xc2x0 C. for 5-10 hours. Using these protecting groups, PO oligonucleotides and other modified oligonucleotides can be synthesized. But in certain instances where modified oligonucleotides are functionalized with base-sensitive groups, the functionalities often get removed while the deprotection is being carried out. Examples of such base-sensitive modified oligonucleotides include, ribonucleoside-containing oligonucleotides, methylphosphotriester oligonucleotides, phosphoramides, etc. In particular, the large-scale synthesis of RNA which is required for the ribozyme-based therapeutic strategies presents special challenges due to two factors. These are, first, 3xe2x80x2-5xe2x80x2to 2xe2x80x2-5xe2x80x2internucleotide chain migration during preparation of nucleoside monomer precursors, during synthesis, and during removal of protecting groups from the RNA, and second, degradation of RNA. Use of classical protecting groups compounds these factors. For successful RNA synthesis, it is essential that the 2xe2x80x2 hydroxyl protecting group remains intact until the final deprotection step and that following its removal, the 2xe2x80x2 hydroxyl group does not attack the vicinal phosphodiester groups and thereby promote cleavage or migration of the internucleotidic linkages. In other applications of oligonucleotides, it is desirable to have oligonucleotides still bound to the solid support. Such completely deprotected oligonucleotides still bound to the solid support can be useful in a variety of applications such as those involving isolation of transcription factors and other factors or elements that interact with oligonucleotides. They are also useful for solid-phase PCR, investigation into nucleic acid protein interactions by, for example, NMR, creation and use of combinatorial libraries, screening of nucleic acid libraries, and solid support based hybridization probes (analogous to Southern and Northern blotting protocols). Creating such a support bound, deprotected oligonucleotide would be greatly aided by having a protecting group that could be removed by mild conditions that would not cleave the oligonucleotide from the support.
There is, therefore, a need for methods for oligonucleotide synthesis that allow for deprotection of the oligonucleotide under more mild conditions than existing methods. There is further a need for nucleoside synthons having new base protecting groups that are stable under oligonucleotide synthesis conditions, but which can be removed under more mild conditions than existing protecting groups.
The invention provides new methods for synthesizing oligonucleotides that allow for deprotection of the oligonucleotide under more mild conditions than existing methods. The invention further provides a nucleoside base protecting group that is stable under oligonucleotide synthesis conditions, but which can be removed under more mild conditions than existing protecting groups, as well as nucleoside synthons having such base protecting groups.
In a first aspect, the invention provides a novel nucleoside base protecting group having the general structure I: 
wherein n1, n2 and n3 are each independently 0-10, wherein a, b, c, d and e are each independently hydrogen, carbon or nitrogen, and wherein the ring structure bearing substituent R3 shown may be aromatic or heterocyclic, wherein the nitrogen displayed is the protected amino moiety of the nucleoside base, wherein R1, R2 and R3 are independently hydrogen, or an alkyl, aryl, aralkyl, ether, hydroxy, nitrile, nitro, ester, carboxyl, or aldehyde group, and wherein dotted lines represent alternative exocyclic or endocyclic double bonds. In a preferred embodiment, a is hydrogen when n1 is 0 and is carbon or nitrogen when n1 is 1-10, b is hydrogen when n1 and n2 are both 0 and is carbon or nitrogen when either or both n1 and n2 are 1-10, c is hydrogen when n2 is 0 and is carbon or nitrogen when n2 is 1-10, and e is hydrogen when n2 is 0 and is carbon or nitrogen when n3 is 1-10. In a particularly preferred embodiment, compound I has n1, n2 and n3 values of 0, and a, b, c, d and e are each hydrogen, and the protecting group takes the form N-pent-4-enoyl, i.e., CH2xe2x95x90CH(CH2)2COxe2x80x94(II). Compounds I and II protect the nucleoside base amino moieties by forming amide linkages, as in: 
where the nitrogen displayed is the protected amino moiety of the base B.
Base protecting group I and the preferred embodiment II are particularly advantageously used because such protecting group can be removed chemoselectively by treatment with a chemoselective removing agent. Thus, in a second aspect, the invention provides a method for synthesizing oligonucleotides that allows for removal of base protecting groups under more mild conditions than existing methods. This new method comprises sequentially coupling nucleoside synthons having base protecting groups according to the invention to produce a base-protected oligonucleotide, followed by deprotection using a chemoselective removing agent. The method according to the invention can utilize any known or otherwise suitable oligonucleotide synthesis chemistry, including the well known H-phosphonate and phosphoramidite chemistries.
The use of this new method provides numerous advantages. For example the method""s mild procedure for removing the protecting group without affecting the integrity of other functionalities present in the oligonucleotide makes it possible to prepare novel analogs of oligonucleotides such as ribonucleoside-containing oligonucleotides, alkylphosphotriesters, certain base-sensitive phosphoramidate and other base-sensitive oligonucleotides. Besides being able to synthesize oligonucleotides bearing xe2x80x9csensitivexe2x80x9d functionalities, it can also be used in the routine synthesis of various oligonucleotides as in case of the conventional protecting groups. In addition, this new method allows for synthesis of oligonucleotides still bound to any type of solid support. Where an unprotected, support-bound oligonucleotide is desired, the full length support-bound oligonucleotide can have its internucleoside linkages oxidized, followed by contacting the oligonucleotide with a chemoselective removing agent to cleave the base protecting group.
A preferred use of this aspect of the invention is in the synthesis of RNA. Preferably, such synthesis employs a phosphoramidite, H-phosphonate or phosphotriester nucleoside monomer synthon having novel protecting groups according to the invention on the nucleoside base, as well as on the 2xe2x80x2 hydroxyl of the nucleoside sugar.